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Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization


Gram-negative and Gram-positive bacteria release a variety of membrane vesicles through different formation routes. Knowledge of the structure, molecular cargo and function of bacterial extracellular vesicles (BEVs) is primarily obtained from bacteria cultured in laboratory conditions. BEVs in human body fluids have been less thoroughly investigated most probably due to the methodological challenges in separating BEVs from their matrix and host-derived eukaryotic extracellular vesicles (EEVs) such as exosomes and microvesicles. Here, we present a step-by-step procedure to separate and characterize BEVs from human body fluids. BEVs are separated through the orthogonal implementation of ultrafiltration, size-exclusion chromatography (SEC) and density-gradient centrifugation. Size separates BEVs from bacteria, flagella and cell debris in stool; and blood cells, high density lipoproteins (HDLs) and soluble proteins in blood. Density separates BEVs from fibers, protein aggregates and EEVs in stool; and low-density lipoproteins (LDLs), very-low-density lipoproteins (VLDLs), chylomicrons, protein aggregates and EEVs in blood. The procedure is label free, maintains the integrity of BEVs and ensures reproducibility through the use of automated liquid handlers. Post-separation BEVs are characterized using orthogonal biochemical endotoxin and Toll-like receptor-based reporter assays in combination with proteomics, electron microscopy and nanoparticle tracking analysis (NTA) to evaluate BEV quality, abundance, structure and molecular cargo. Separation and characterization of BEVs from body fluids can be done within 72 h, is compatible with EEV analysis and can be readily adopted by researchers experienced in basic molecular biology and extracellular vesicle analysis. We anticipate that this protocol will expand our knowledge on the biological heterogeneity, molecular cargo and function of BEVs in human body fluids and steer the development of laboratory research tools and clinical diagnostic kits.

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Fig. 1: Schematic size and density indication of BEVs compared to contaminants present in stool and/or blood plasma.
Fig. 2: Illustrative overview of the BEV separation protocol.
Fig. 3: Possibilities for characterization of BEVs in relation to their concentration.
Fig. 4: Optimization of different steps of the orthogonal separation methods.

Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The computer code used for the robot-assisted steps of the protocol (Steps 20A and 27A) is available from the corresponding author upon reasonable request.


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This work was supported by Krediet aan Navorsers and a fellowship (A.H.) from Research Foundation Flanders (FWO) and Concerted Research Action from Ghent University.

Author information




J.T., O.D.W. and A.H. acquired and analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to An Hendrix.

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Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Cherie Blenkiron and Masanori Toyofuku for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Tulkens, J. et al. Gut (2018):

EV-TRACK Consortium, Van Deun, J. et al. Nat. Methods 14, 228–232 (2017):

Van Deun, J. et al. J. Extracell. Vesicles 3, 24858 (2014):

Integrated supplementary information

Supplementary Fig. 1 BEV as potent stimulators of PBMC.

After stimulation of PBMC with BEVs, immediate release of chemo- and cytokines can be observed after performing Luminex assay. As proof of concept, we show here IL-8 and TNF-α concentration as a function of time after BEV stimulation. In this study, collection of blood and stool samples was according to Ethical Committee of Ghent University Hospital and in accordance to relevant guidelines. The patients provided informed written consent. PBMC, peripheral blood mononuclear cells.

Supplementary Fig. 2 Evaluation of the performance of size exclusion chromatography.

At least 1×1010 EcN BEVs are spiked in 2 ml plasma or PBS and SEC is performed. LPS activity of the collected SEC fractions is measured by LAL assay. A first peak should be visible in SEC fractions 4-6 when the technique is performed correctly. Western blot (using anti-LPS (1:1,000) or anti-OmpA (1:5,000) antibodies) can further validate these results. Figure adapted from Tulkens et al.6. In this study, collection of blood samples was according to Ethical Committee of Ghent University Hospital and in accordance to relevant guidelines. The patients provided informed written consent. EcN, Escherichia coli Nissle 1917.

Supplementary information

Supplementary Information

Supplementary Figures 1 and 2

Reporting Summary

Supplementary Video 1

Robot-assisted preparation of density gradients.

Supplementary Video 2

Loading of a sample on a size-exclusion chromatography column.

Supplementary Video 3

Non-stimulated PBMCs recorded using a live-cell analysis system.

Supplementary Video 4

BEV-induced PBMC stimulation recorded using a live-cell analysis system.

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Tulkens, J., De Wever, O. & Hendrix, A. Analyzing bacterial extracellular vesicles in human body fluids by orthogonal biophysical separation and biochemical characterization. Nat Protoc 15, 40–67 (2020).

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